Transcriptomics: Lecture 2
Frontiers of Biotechnology: Bioinformatics and Systems Modelling
The University of Adelaide
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Welcome To Country
I’d like to acknowledge the Kaurna people as the traditional owners and custodians of the land we know today as the Adelaide Plains, where I live & work.
I also acknowledge the deep feelings of attachment and relationship of the Kaurna people to their place.
I pay my respects to the cultural authority of Aboriginal and Torres Strait Islander peoples from other areas of Australia, and pay our respects to Elders past, present and emerging, and acknowledge any Aboriginal Australians who may be with us today
RNA-Seq
RNA Sequencing
According to Wang, Gerstein, and Snyder (2009)
RNA-Seq, also called RNA sequencing, is a particular technology-based sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome.
RNA Sequencing
- Microarrays are still published regularly
- Also used extensively for methylation
- RNA sequencing is now the dominant technology
- Strong improvement for:
- transcript-level resolution
- un-annotated genes
- allelic bias
- genomic variants
The RNA Population Of a Eukaryotic Cell
The Key Steps
- Focus from here on will be sequencing mRNA using short reads
- Library Preparation
- RNA Quality assessment
- Selecting target molecules
- Adding sequencing primers
- Sequencing
- Aligmnent + Quantitation
- DE Gene Detection
- Downstream Analysis
- (Optional) Nobel Prize
RNA Selection
- rRNA makes up about 80% of cellular RNA \(\rightarrow\) not of general interest
- tRNA ~15% + mRNA ~5% cellular RNA1
- Select for poly-adenylated RNA using oligo-dT-based methods
- Only extracts intact mRNA with a polyA tail (includes some ncRNA)
Library Preparation
- RNA is then fragmented and size selected (200-300nt)
- Very short transcripts always lost during this step
- cDNA produced
- Sequencing adapters added
- Many adapters now contain Unique Molecular Identifiers (UMI)
- Helps identify PCR duplicates
References
Footnotes
https://bionumbers.hms.harvard.edu/bionumber.aspx?s=n&v=5&id=100264↩︎